Development of Radiolabeled Ligands Targeting the Glutamate Binding Site of the N-Methyl-d-aspartate Receptor as Potential Imaging Agents for Brain

Article abstract of DOI:10.1021/acs.jmedchem.6b01344

Abnormal activity of various N-methyl-d-aspartate receptor (NMDAR) subtypes has been implicated in a wide variety of neurological disorders such as Alzheimer's disease, schizophrenia, and epilepsy. Imaging agents for PET and SPECT that target NMDARs in a subtype-selective fashion may enable better characterization of those disorders and enhance drug development. On the basis of a pyrazoline derivative that demonstrated neuroprotective effects in vivo, we synthesized a series of para-substituted analogues and measured their affinities to various NMDAR subtypes. Compounds 4a-c and 4e showed greater, nanomolar affinity for the GluN1/2A subtype versus GluN1/2B. Dicarbomethoxy (pro-drug) analogues of [^124/125I]4d and [¹¹C]4e (i.e., [^124/125I]11d and [¹¹C]11e) were generated and tested for NMDAR binding specificity in ex vivo autoradiography and brain biodistribution studies. Although NMDAR-specific binding could be demonstrated for [¹²⁵I]11d and [¹¹C]11e through autoradiography and biodistribution studies, imaging of neither [¹²⁴I]11d nor [¹¹C]11e could demonstrate brain penetration sufficient for detection by PET.

Full text of DOI:10.1021/acs.jmedchem.6b01344

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Article
Development of radiolabeled ligands targeting the glutamate binding site
of the N-methyl-D-aspartate receptor as potential imaging agents for brain
Lucia Tamborini, Ying Chen, Catherine A. Foss, Andrea Pinto, Andrew G. Horti, Stephen F.
Traynelis, Carlo De Micheli, Ronnie C Mease, Kasper B Hansen, Paola Conti, and Martin G Pomper
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01344 • Publication Date (Web): 21 Nov 2016
Downloaded from http://pubs.acs.org on November 25, 2016
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Development of radiolabeled ligands targeting the
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glutamate binding site of the Nꢀmethylꢀ ꢀaspartate
D
receptor as potential imaging agents for brain
Lucia Tamborini,a Ying Chen,b Catherine A. Foss,b Andrea Pinto,a Andrew G. Horti,b Stephen F.
Traynelis,c Carlo De Micheli,a Ronnie C. Mease,b Kasper B. Hansen,d*
Paola Conti,a* Martin G. Pomperb*
aUniversità degli Studi di Milano, Department of Pharmaceutical Sciences, Via Mangiagalli 25,
20133, Milano, Italy; bRussell H. Morgan Department of Radiology and Radiological Science,
Johns Hopkins Medical School, Baltimore, MD, USA; cDepartment of Pharmacology, Emory
University School of Medicine, Atlanta, GA, USA; dDepartment of Biomedical &
Pharmaceutical Sciences, University of Montana, 32 Campus Drive, Missoula, MT 59812
KEYWORDS: NMDAR, glutamate, pyrazoline, PET, autoradiography
ABSTRACT
Abnormal activity of various NꢀmethylꢀDꢀaspartate receptor (NMDAR) subtypes has been
implicated in a wide variety of neurological disorders such as Alzheimer’s disease, schizophrenia
and epilepsy. Imaging agents for PET and SPECT that target NMDARs in a subtypeꢀselective
fashion may enable better characterization of those disorders and enhance drug development.
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Based on a pyrazoline derivative that demonstrated neuroprotective effects in vivo, we
synthesized a series of paraꢀsubstituted analogs and measured their affinities to various NMDAR
subtypes. Compounds 4aꢀc and 4e showed greater, nanomolar affinity for the GluN1/2A subtype
versus GluN1/2B. Diꢀcarbomethoxy (proꢀdrug) analogs of [124/125I]4d and [11C]4e (i.e.,
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[
124/125I]11d and [11C]11e) were generated and tested for NMDAR binding specificity in ex vivo
autoradiography and brain biodistribution studies. Although NMDARꢀspecific binding could be
demonstrated for [125I]11d and [11C]11e through autoradiography and biodistribution studies,
imaging of neither [124I]11d nor [11C]11e could demonstrate brain penetration sufficient for
detection by PET.
Introduction
Glutamateꢀactivated receptors belong to two families: ionotropic glutamate receptors (iGluRs)
and metabotropic glutamate receptors (mGluRs). Within the class of ionotropic glutamate
receptors, NꢀmethylꢀDꢀaspartate receptors (NMDARs) have been widely investigated for their
involvement in neurodegenerative processes.1 The majority of NMDA receptors are
heterotetrameric assemblies of two glycineꢀbinding GluN1 subunits and two glutamate binding
GluN2 subunits, of which four types exist (GluN2A, B, C, D).2 The various GluN2 subunits
show differential temporal and spatial distribution in the central nervous system (CNS), and
provide an opportunity to develop regionꢀspecific pharmacological regulation of NMDA receptor
function using compounds selective for one individual GluN2 subunit.3
Unlike dopaminergic and serotonergic transmission, glutamatergic transmission has proven to
be an elusive target for brain imaging, although several promising agents exist.4,5 Positron
emission tomography (PET) and single photon emission computed tomography (SPECT)
imaging agents for glutamate receptors (GluRs) could be powerful tools for the evaluation of
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excitatory neurotransmission, the study of the pathophysiology of related neurological disorders,
and the quantification of GluR drug occupancy in vivo. The strategy for the development of PET
and SPECT probes for NMDA receptors involves the structural modification of NMDA receptor
antagonists, including channel blockers, e.g. [11C]ketamine, [18F]fluoromemanthine and Nꢀ(1ꢀ
naphthyl)ꢀ͈ꢀ(3ꢀ[123I]iodophenyl)ꢀ͈ꢀmethylguanidine ([123I]CNSꢀ1261, 1),6 glycine site
antagonists, e.g. 3ꢀ[3ꢀ(4ꢀ[11C]methoxybenzyl)phenyl]ꢀ4ꢀhydroxyꢀ7ꢀchloroquinolinꢀ2(1H)ꢀone
([11C]Lꢀ703,717),6 and GluN2B negative modulators, e.g. compound 2,6 (Figure 1).
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11CH3
CH3
N
NH2
H
NH
O
N
123I
18F
Cl
NH
1, [123I]CNS-1261
[18F]fluoromemanthine
[11C]ketamine
11CH3
O
OH
OH
OH
O11CH3
N
Cl
N
H
O
CH3
HO
[11C]L-703,717
2
Figure 1. Structures of imaging agents for NMDA receptors.
For example, 1 is a channelꢀbinding NMDA receptor radioligand for SPECT that has been used
to study NMDA receptor function in vivo in humans.7 While of high affinity and having been
tested in patients with schizophrenia, changes in 1 signal noted in affected individuals relative to
controls, including those on clozapine, were small.8 The results obtained with 1 proved
sufficiently encouraging to pursue other compounds of its class for PET.5 Nevertheless, most
competitive antagonists of the glutamate binding site have shown low selectivity for NMDARs
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and poor bloodꢀbrain barrier (BBB) permeability, and at present there are no validated
radioligands interacting with this region of the NMDARs.
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In the last decade, we developed several new competitive NMDA receptor antagonists, as
potential neuroprotective agents.9 In particular, compound 3 (Figure 2) was highlighted for its
promising pharmacological activity as a neuroprotective agent.10 In binding assays, compound
(5S,αR)ꢀ3 showed nanomolar affinity for native receptors. Furthermore, preliminary evaluation
of GluN2 subunitꢀselectivity suggested that the racemic mixture (±)ꢀ3 displayed a preference for
inhibition of NMDA receptors containing GluN2A or GluN2B subunits compared to receptors
with GluN2C or GluN2D subunits.10
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H2N
H2N
HOOC
HOOC
COOH
COOH
R
F
N
N
N
a
b
c
d
e
N
Cl
Br
I
CH3
R
(5S, αR)-4a-e
(5S, αR)-3
Figure 2. Structures of model and target compounds
Despite being a charged molecule, (±)ꢀ3 displayed anticonvulsant activity after intraꢀperitoneal
(ip) administration to mice, indicating the capacity to cross the intact BBB at the doses
employed.10 Therefore, (5S,αR)ꢀ3 appeared to us as a suitable tool to generate radiolabeled
analogues for PET (or SPECT) imaging within the CNS.
Considering the molecular structure of (5S,αR)ꢀ3, we identified the para position of the
aromatic ring as a suitable position for the insertion of a radiolabeled atom or group. We
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synthesized and pharmacologically tested several pꢀsubstitutedꢀanalogs, i.e. (5S,αR) 4aꢀe (Figure
2), to measure how para substitution would affect the binding properties in terms of affinity and
subtype selectivity. The para substituents included either a halogen (F, Br, I, compounds 4a, c,
d) or a methyl group (compound 4e), that could be subsequently replaced by their radioactive
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counterpart, namely, F, Br, 124/125I, or C. For a more thorough description of the structureꢀ
activity relationships (SARs), we included in this series also the pꢀCl derivative (compound 4b).
The clogD and polar surface area (PSA) values of compounds 4aꢀe were calculated as shown in
Table 1. The negative clogD and high PSA values caution that, although the (5S,αR)ꢀ3 possesses
CNS activity, 4aꢀe may not have sufficient BBB penetration when administered at the radiotracer
level. Converting 4aꢀe to the prodrugs 11aꢀe, containing biologically cleavable methyl esters
groups, significantly increased the clogD to positive values and also decreased PSA values,
which may improve BBB penetration.
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Table 1. Calculated CLogD and Polar Surface Areas
Compd. CLogD
(pH 7.4)
PSA
ꢀ3.9
ꢀ3.4
ꢀ3.2
ꢀ3.0
ꢀ3.8
0.1
116.2
116.2
116.2
116.2
116.2
94.2
4a
4b
4c
4d
4e
11a
11b
11c
11d
0.7
94.2
0.9
94.2
1.1
94.2
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0.3
94.2
11e
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aACD/LogD Software, Advanced Chemistry Development, ACD/Labs Release 9.0, Product
Version 9.1
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Compounds 4d, 11d (prodrug of 4d) and 11e (prodrug of 4e) were then prepared in radioactive
form and submitted for biological investigation.
Results and discussion
Chemistry
Chloroꢀhydrazones 7a-e were prepared by reacting the pꢀsubstituted anilines 5a-e with methyl
2ꢀchloroꢀ3ꢀoxobutanoate 6, in the presence of sodium nitrite, 6 N HCl, sodium acetate and
MeOH at 0 °C.11 Chloroꢀhydrazones 7a-e were treated with base to generate in situ the
corresponding Nꢀsubstitutedꢀmethoxycarbonylformonitrilimines that underwent 1,3ꢀdipolar
cycloaddition with suitably protected allylglycine 8 (Scheme 1). The cycloaddition reaction
1
produced a mixture of stereoisomers 9 and 10 in an approximate 1:1 ratio, as estimated by H
NMR spectroscopy. Chromatographic separation of the two stereoisomers was unsuccessful at
this stage; therefore, the mixture was treated with a 30% solution of trifluoroacetic acid (TFA) in
dichloromethane to remove the NꢀBoc protecting group. The couples of amines 11a-e and 12a-e
were easily separated by flashꢀchromatography. The desired stereoisomers 11a-e were then
converted into the final amino acids 4a-e through a threeꢀstep protection/deprotection strategy
including a) NꢀBoc protection, b) hydrolysis of the two ester functions, and c) NꢀBoc
deprotection, using standard procedures.
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H3COOC Cl
R
NH2
H3COOC Cl
N
NaNO2, HCl 6N, AcONa
a
b
c
d
e
F
NH
Cl
Br
I
+
MeOH, 0°C
ref. 11
H3C
O
9
R
CH3
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3a-e
6
R
7 a-e
HOOC
d
H3COOC
NH2
COOH
NH2
COOCH3
N
N
N
R
N
R
H3COOC Cl
N
H3COOC
NHBoc
NHBoc
a
b
N
+
NH
COOCH3
N
4 a-e
11a-e
9a-e
COOCH3
c
8
H3COOC
R
NH2
R
a-e
N
7 a-e
9
COOCH3
N
R
10 a-e
12 a-e
Scheme 1. a) NaHCO3, EtOAc, ꢁ, 70ꢀ90% yield; b) i) 30% TFA, CH2Cl2, 78ꢀ87% yield; ii) flash
chromatography; c) Boc2O, TEA, CH2Cl2, quantitative yield; d) i) 1N NaOH, MeOH; ii) 30%
TFA, CH2Cl2, 58ꢀ63% yield after 2 steps.
Pharmacology
Final amino acids 4aꢀe were evaluated using twoꢀelectrode voltageꢀclamp electrophysiology at
recombinant NMDA receptors expressed in Xenopus oocytes (Supplemental Figure 1 and Table
2).
Table 2. IC50 values for inhibition of current responses activated by 100 µM glycine and Lꢀ
glutamate in oocytes expressing recombinant rat GluN1/2A, GluN1/2B, GluN1/2C, and
GluN1/2D receptors. 10 µM Lꢀglutamate was used for GluN1/2A, 3 µM Lꢀglutamate for
GluN1/2B and GluN1/2C, and 1 µM Lꢀglutamate for GluN1/2D receptors. Ki values were
estimated using the ChengꢀPrusoff relationship with the following Lꢀglutamate EC50 values:
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GluN1/2A 3.8 µM, GluN1/2B 1.9 µM, GluN1/2C 1.0 µM, and GluN1/2D 0.45 µM.12,13 Hill
slopes for the concentrationꢀinhibition curves were 1.1 to 1.4, and 5ꢀ8 oocytes were recorded for
each IC50 value using twoꢀelectrode voltageꢀclamp electrophysiology.
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Cpd. GluN1/2A
GluN1/2B
Ki IC50
GluN1/2C
Ki IC50
GluN1/2D
Ki IC50
IC50
(nM)
Est.
(nM)
Est.
(nM)
Est.
(nM)
Est.
(nM)
Ki
(nM)
(nM)
(nM)
320 ±10 87±4
210±40
860±10
280±20
250±20
700±40
690±50
80±15
330±40
110±10
94±7
200±10
920±20
180±10
190±10
560±20
380±20
49±2
390±80
920±20
520±20
630±40
120±30
290±10
160±10
200±10
3
160±10
98±4
43±2
27±1
25±1
250±20
48±3
230±10
44±2
4a
4b
4c
4d
4e
91±4
47±3
910±80
180±10
270±20
250±20
140±10
96±5
1.310±90 110±30
1.420±60 440±20
The estimated Ki values for the model compound (5S,αR)ꢀ3 at GluN1/2A and GluN1/2B
receptor subtypes were 87 nM and 80 nM, respectively. Interestingly, we observed that the
insertion of a substituent in the para position of compound (5S,αR)ꢀ3 gave rise to compounds
endowed with an increased affinity and selectivity for the GluN1/2A subtype versus GluN1/2B.
In detail, by increasing the size of the substituent, the affinity for GluN1/2A increased up to a
certain size (H < F, CH3 < Cl, Br) and then it dropped with larger substituents such as I
(compound 4d). Notably, the best selectivity GluN1/2A over GluN1/2B was obtained with the
insertion of F (compound 4a) or CH3 (compound 4e).
The estimated Ki values for compounds 4a and 4e were 7.6ꢀfold and 5.3ꢀfold higher,
respectively, at GluN1/2B compared to at GluN1/2A (namely, the binding affinities were higher
at GluN1/2A). Insertion of Cl (compound 4b) resulted in an estimated Ki value that was 4.0ꢀfold
higher at GluN1/2B compared to at GluN1/2A. Among glutamate site competitive antagonists
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described to date, [(R)ꢀ[(S)ꢀ1ꢀ(4ꢀbromophenyl)ꢀethylamino]
ꢀ
(2,3ꢀdioxoꢀ 1,2,3,4
ꢀ
5
6
7
8
tetrahydroquinoxalin ꢀ 5 ꢀ yl ) ꢀ methyl]ꢀphosphonic acid (NVPꢀAAM077)14 is generally
considered to have the highest selectivity for GluN1/2A over GluN1/2B. However, its binding
affinity is only modestly 5.2ꢀfold higher for GluN1/2A (15 nM) compared to GluN1/2B (78
nM).15 Thus, the 7.6ꢀfold selectivity of compound 4a for GluN1/2A over GluN1/2B represents a
useful starting point for the development of a PET or SPECT ligand to image GluN1/2A
receptors in the CNS. The 18Fꢀlabeled analog of 4a proved synthetically challenging requiring us
to focus our radiosynthetic efforts on [11C]4e and the corresponding radioꢀiodinated analogs for
SPECT (125I) and PET (124I), [125/124I]4d.
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Radiochemistry
H3COOC
t-BuOOC
t-BuOOC
HOOC
NHBoc
COOCH3
NHBoc
COOt-Bu
NHBoc
COOt-Bu
NH2
COOH
N
a
N
N
N
b
c
N
N
N
N
Br
Br
SnBu3
125I
125I]4d
9c
13
14
[
Scheme 2. a) i) 1N NaOH, MeOH, 95% yield; ii) TBTA, dry THF/CH2Cl2, 42% yield; b)
Bu6Sn2, Pd(PPh3)4, toluene, 55% yield; c) i) AcOH, [125]NaI, NCS, MeOH; ii) TFA/H2O, 54%
radiochemical yield.
The synthesis of the stannyl precursor 14 for the radioligand [125I]4d is shown in Scheme 2.
Aryl bromide 13, prepared starting from 9c, was treated with hexabutylditin in the presence of
tetrakis(triphenylphosphine)palladium in toluene to give 14. Radioiodination of 14 was carried
out by treatment with [125I]NaI in the presence of Nꢀchlorosuccinimide followed by deprotection
with TFA/H2O to give [125I]4d.
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H3COOC
H3COOC
H3COOC
NHBoc
COOCH3
NHBoc
COOCH3
NH2
COOCH3
N
N
a
N
d
N
N
N
SnBu3
11CH3
Br
[
11C]11e
15
c
9c
b
H3COOC
H3COOC
NH2
COOCH3
NH2
COOCH3
N
N
N
N
124I
[
124I]11d
125I
[
125I]11d
Scheme 3. a) Bu6Sn2, Pd(PPh3)4, toluene, 33% yield; b) i) AcOH, [125]NaI, NCS, MeOH,
radiochemical yield 58%; ii) TFA/H2O; c) AcOH, [124]NaI, NCS, MeOH; ii) TFA/H2O,
radiochemical yield 49%; d) i) [11C]CH3I, Pd2(dba)3, P(oꢀtol)3, DMF; ii) TFA/H2O,
radiochemical yield 2ꢀ5%.
The synthesis of the stannyl precursor 15 for the radioligand [125I]11d, [124I]11d and [11C]11e
is shown in Scheme 3. Aryl bromide 9c was treated with hexabutylditin in the presence of
tetrakis(triphenylphosphine)palladium in toluene to give 15. Radioiodination of 15 was carried
out by treatment with [125I]NaI or [124I]NaI in the presence of Nꢀchlorosuccinimide followed by
deprotection with TFA/H2O to give [125I]11d or [124I]11d. [11C]11e was prepared by Stille
coupling reaction of 15 with [11C]methyl iodide followed by deprotection with TFA/H2O.
Autoradiography
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Ex vivo autoradiography studies were conducted in female athymic nude mice with radiolabeled
compounds [125I]4d and prodrug [125I]11d to qualitatively assess both the ability to cross the
BBB as well as brain subregion selectivity. Each mouse was injected with radiotracer, allowed to
undergo the indicated uptake time (Figure 3), sacrificed and then sections were cut and exposed
to xꢀray film. Compound [125I]4d displayed no evidence of brain uptake (Figure 3A) while
prodrug compound [125I]11d displayed brain regionꢀselective uptake (Figure 3B), particularly in
cortex, hippocampus, thalamus (Supplemental Figure 2) and cerebellum, with absence of uptake
in caudateꢀputamen, which is consistent with the distribution of GluN1/2A subtype receptor
mRNA expression.16 Brain uptake of [125I]11d exhibited highꢀcontrast and regionꢀspecific
retention but was low in absolute value as estimated by exposure time.
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Figure 3. In vivo and ex vivo brain uptake and distribution of [125I]4d and [125I]11d prodrug in
mice. A. Ex vivo autoradiography using [125I]4d in serial brain sections following a 60 min
conscious uptake. No evidence of radiotracer penetration into brain tissue is apparent. B. Ex
vivo autoradiography using [125I]11d prodrug in serial brain sections following a 40 min
conscious uptake. Evidence of selective uptake in GluN2A subunitꢀrich regions is apparent
including cortex (Ctx), hippocampus (H) and cerebellum (Cer). C. Ex vivo biodistribution of
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[124I]11d in mice reflecting a 75 min uptake period confirms low overall brain uptake in the
indicated tissue regions, indicating unsuitability for in vivo imaging.
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Biodistribution
Ex vivo biodistribution in CDꢀ1 mice was undertaken with prodrug compounds [124I]11d and
[11C]11e to ascertain and quantify regionꢀselective uptake. Compound [124I]11d was injected into
mice and allowed to undergo a 75 min biological uptake period to maximize in vivo prodrug
cleavage and receptor binding. Figure 3C shows a graph of those uptake values as percentage of
injected dose per gram of wet tissue (% ID/g) in selected regions. Uptake across regions was low
and never exceeded 1% ID/g. In vivo PET imaging with this compound was also undertaken
through 90 min and verified that brain uptake is below the threshold of detection for this
modality (Supplemental Data).
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Figure 4. Ex vivo brain biodistribution of [11C]11e prodrug in mice. A. Mice were injected with
[11C]11e and sacrificed at the indicated uptake times. The pattern of uptake in indicated brain
tissues shows increasing radiotracer uptake over time in all regions except cerebellum and the
remaining brain tissue following dissection. Significant radiotracer uptake was observed in
frontal cortex (P = 0.004) and superior colliculus (P = 0.04) between 45 and 60 min of uptake. B.
Autoblockade at 60 min after radiotracer injection compared with radiotracer alone at 60 and 75
min postꢀinjection. Blockade was observed in cerebellum (85%), olfactory bulb (88%),
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hippocampus (98%), frontal cortex (80%), cortex (60%), brainstem (77%), thalamus (45%) and
in the remaining tissue (71%). Radiotracerꢀonly uptake increased from 60ꢀ75 min in the same
regions as in (A) where significance was only reached in striatum between 60 and 75 min of
uptake (P = 0.009). Note: Specific radiotracer activity in (A) was 1,400 Ci/mmol while in (B) it
was 11,500 Ci/mmol at the time of injection, which may account for differences in absolute %
ID/g values.
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Dimethyl ester [11C]11e, for which GluN1/2A affinity of the free acid analog 4e was found to be
higher than that for 4d, was also assayed by ex vivo biodistribution. Figure 4 shows the graph of
uptake values from 15 min through 60 min of uptake (Figure 4A) as well as autoblockade at 60
min (Figure 4B) to verify radiotracer specificity. Figure 4A demonstrates radiotracer washꢀin at
15 min followed by reduction at 30 min and then steady accumulation through 60 min,
suggesting in vivo cleavage of the methyl esters and subsequent receptor binding over time.
Figure 4B shows the effect of coꢀadministration of 4.5 mg/kg of unlabeled compound 11e with
radiotracer at 60 min of uptake time as well as increasing uptake in radiotracerꢀonly mice from
60 to 75 min, continuing the trend seen in Figure 4A.
Conclusion
Here we report the development of radiolabeled ligands [125I]4d, [124/125I]11d and [11C]11e for
in vivo targeting of NMDA receptors. [125I]4d was not able to cross BBB when administered
intravenously as a radiotracer. Applying a prodrug approach, both [124/125I]11d and [11C]11e,
containing cleavable methyl ester groups, did demonstrate BBB penetration following
intravenous administration and exhibited differentially selective brain subꢀregional binding.
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124/125I]11d displayed an autoradiographic uptake profile consistent with the distribution of
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GluN2A subunits16 while ex vivo biodistribution of [11C]11e suggested binding to both
GluN1/2B and GluN1/2C, which accounts for both significant measured uptake in superior
colliculus (GluN2C profile) and caudateꢀputamen (GluN2B profile). Compounds 11d and 11e
differ only in the radiolabeled atom present in the same para position on the aryl ring where 11d
contains a large iodine atom and 11e contains a smaller, less lipophilic methyl group. These
differences may account for different murine in vivo GluN2 subunit binding selectivity,
contrasting with the trends measured in Xenopus oocyte extracts in Table 2. It is also possible
that the murine brain contains disparately distributed methyl esterase activity and/or each analog
has a significantly different affinity for the esterase, efficient cleavage of which is necessary to
convert prodrug analogs into the dicarboxylic acid species necessary to bind the targeted
receptors (Supplemental Table 1).
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Aside from the ex vivo evidence showing selective brain uptake for 11d and 11e, in vivo PETꢀ
CT imaging with [124I]11d revealed no observable brain uptake (Supplemental Data). That was
not unexpected when measured uptake values from ex vivo biodistribution studies revealed %
ID/g values consistently below 1% ([124I]11d, Figure 3C) or below 1.5% ID/g ([11C]11e, Figure
4A, B). Although we previously showed that 3 could demonstrate neuroprotective properties,10
further structureꢀactivity relationships expanding upon this scaffold are necessary to identify
compounds with sufficient affinity and suitable lipophilicity to serve as imaging agents for
NMDARs in CNS. SAR studies will also be aimed at improving even further the specificity for
the different subtypes of NMDA receptors. A potential utility for freeꢀacids [124I]4d and [11C]4e
would include imaging of NMDARs in peripheral neuroendocrine and neuroendocrineꢀlike
tumors, including melanomas, breast, smallꢀcell lung cancer and castrateꢀresistant prostate
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cancers.17 These free acids possess high affinity for their receptor subtypes (Table 2) and
favorable clogD values (Table 1) for peripheral imaging.
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Experimental
Solvents and chemicals purchased from commercial sources were of analytical grade or better
and used without further purification. 1H NMR and 13C NMR spectra were recorded on a Bruker
Ultrashield 400 MHz or on a Varian Mercury 300 (300 MHz) spectrometer. Chemical shifts (δ)
are expressed in ppm, and coupling constants (J) are expressed in Hz. Rotary power
determinations were carried out using a Jasco Pꢀ1010 spectropolarimeter, coupled with a Haake
N3ꢀB thermostat. TLC analyses were performed on commercial silica gel 60 F254 aluminum
sheets; spots were further evidenced by spraying with a dilute alkaline potassium permanganate
solution. Melting points were determined on a model B 540 Büchi apparatus and are uncorrected.
MS analyses were performed on a Varian 320ꢀMS triple quadrupole mass spectrometer with ESI
source. Microanalyses (C, H, N) of new compounds were within ± 0.4 % of theoretical values.
Chloroꢀhydrazones 7a-e have been synthesized following a literature procedure.11 HPLC
purification of radiolabeled compounds was performed on Varian Prostar systems (Palo Alto,
CA), equipped with a Varian ProStar 325 UV–Vis variable wavelength detector and a Bioscan
Flowꢀcount inꢀline radioactivity detector (Washington, DC). The specific radioactivity was
calculated as the ratio of the radioactivity eluting at the retention time of product during the
preparative HPLC purification to the mass corresponding to the area under the curve of the UV
absorption.
Pharmacological studies
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Two-electrode voltage-clamp recordings
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Rat cDNAs for GluN1ꢀ1a (GenBank IDs U11418 and U08261), GluN2A (D13211), GluN2B
(U11419), GluN2C (M91563), and GluN2D (L31611) were provided by Drs. S. Heinemann
(Salk Institute, La Jolla, CA), S. Nakanishi (Kyoto University, Kyoto, Japan), and P. Seeburg
(University of Heidelberg, Heidelberg, Germany). The cDNA encoding rat GluN2B was
modified without changing the amino acid sequence to remove a T7 RNA polymerase
termination site located in the intracellular Cꢀterminal domain as previously described.18 For
expression of NMDA receptors, cRNA for rat GluN1 (GluN1ꢀ1a splice variant), GluN2A,
GluN2B, GluN2C, and GluN2D were prepared and injected into Xenopus oocytes as previously
described.19 The oocytes were surgically removed from mature female Xenopus laevis and
provided by Xenopus1 (Dexter, MI). Recordings were performed 2ꢀ4 days after injection using
twoꢀelectrode voltageꢀclamp electrophysiology in extracellular solution containing 115 mM
NaCl, 2.5 mM KCl, 1.9 mM BaCl2, and 10 mM HEPES (pH 7.6). The membrane potential was
clamped at −40 mV. During recordings, 100 µM glycine was included in all agonist and/or
antagonist applications. Electrophysiological data were acquired and analyzed as previously
described.19
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Native NMDA receptor binding assays
Affinities for native NMDA receptors in rat cortical synaptosomes were determined using 2 nM
[3H]CGP 39653, as previously described.20
Chemistry
General procedure for the synthesis of compound 4aꢀe.
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a) Derivative 9a-e (1.20 mmol, 1.0 eq.) was dissolved in MeOH (3.6 mL) and treated with 1 N
aqueous NaOH (3.6 mL, 3.0 eq.). The disappearance of the starting material was monitored by
TLC (cyclohexane/EtOAc 8:2). After evaporation of MeOH, the aqueous layer was washed with
Et2O (1 × 5 mL), made acidic with 2 N aqueous HCl and extracted with EtOAc (3 × 5 mL). The
organic layer was dried over anhydrous Na2SO4 and, after evaporation of the solvent, the diacid
derivative was obtained as a yellow solid. b) The diacid derivative (1.18 mmol, 1.0 eq.) was
treated with a 30% CH2Cl2 solution of trifluoroacetic acid (11.8 mmol, 10 eq.) at 0 °C. The
solution was stirred at room temperature for 3 h and the reaction was followed by TLC
(CH2Cl2/MeOH 9:1 + 1% AcOH). The volatiles were removed under reduced pressure and the
residue was crystallized to yield the final aminoacid 4aꢀe.
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(S)-5-((R)-2-Amino-2-carboxyethyl)-1-(4-fluorophenyl)-4,5-dihydro-1H-pyrazole-3-
carboxylic acid (4a). Yield: 60%; yellow solid; m.p. = dec. T > 145 °C; Rf = 0.53
(BuOH/H2O/AcOH 4:2:1); [α]D20 = + 220.00 (c = 0.25; H2O); 1H NMR (300 MHZ, DMSOꢀd6):
1.75–1.85 (1H, m); 1.98–2.10 (1H, m); 2.98 (1H, dd, J = 3.8, 17.8); 3.20 (1H, dd, J = 12.2, 17.8);
3.62–3.75 (1H, m); 4.62–4.78 (1H, m); 7.08–7.25 (4H, m); 13C NMR (75 MHz, DMSOꢀd6):
32.69; 37.35; 50.78; 58.79; 116.46; 116.49 (d, J = 22.2); 139.35; 141.30; 157.67 (d, J = 236);
164.39; 170.89; [M+H]+ = 296.1; anal. calcd for C13H14FN3O4: C, 52.88; H, 4.78; N, 14.23;
found: C, 53.02; H, 4.90; N, 14.11.
(S)-5-((R)-2-Amino-2-carboxyethyl)-1-(4-chlorophenyl)-4,5-dihydro-1H-pyrazole-3-
carboxylic acid (4b). Yield: 63%; yellow solid; m.p. = dec. T > 175 °C; Rf = 0.58
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d6): 1.75–1.85 (1H, m); 1.95–2.10 (1H, m); 3.00 (1H, dd, J = 3.9, 18.3); 3.10 (1H, dd, J = 11.7,
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18.3); 3.60–3.70 (1H, m); 4.60–4.78 (1H, m); 7.20 (2H, d, J = 7.8); 7.35 (2H, d, J = 7.8); C
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NMR (75 MHz, DMSOꢀd6): 32.70; 37.38; 50.87; 58.49; 116.36; 124.79; 129.69; 141.62;
142.35; 164.41; 170.70; [M+H]+ = 312.1; anal. calcd for C13H14ClN3O4: C, 50.09; H, 4.53; N,
13.48; found: C, 50.30; H, 4.62; N, 13.30.
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(S)-5-((R)-2-amino-2-carboxyethyl)-1-(4-bromophenyl)-4,5-dihydro-1H-pyrazole-3-
carboxylic acid (4c). Yield: 60%; yellow solid; m.p. = dec. T > 167 °C; Rf = 0.43
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(BuOH/H2O/AcOH 4:2:1); [α]D = + 285.00 (c = 0.20; DMSO); H NMR (300 MHZ, DMSOꢀ
d6): 1.70–1.85 (1H, m); 1.92–2.02 (1H, m); 2.99 (1H, dd, J = 4.1, 18.1); 3.19 (1H, dd, J = 11.6,
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18.1); 3.55–3.65 (1H, m); 4.60–4.75 (1H, m); 7.15 (2H, d, J = 9.1); 7.41 (2H, d, J = 9.1); C
NMR (75 MHz, DMSOꢀd6): 32.74; 37.45; 50.94; 58.51; 112.65; 116.87; 132.55; 141.95;
142.26; 164.29; 170.57; [M+H]+ = 355.8; anal. calcd for C13H14BrN3O4: C, 43.84; H, 3.96; N,
11.80; found: C, 43.67; H, 3.84; N, 11.92.
(S)-5-((R)-2-Amino-2-carboxyethyl)-1-(4-iodophenyl)-4,5-dihydro-1H-pyrazole-3-
carboxylic acid (4d). Yield: 58%; yellow solid; m.p. = dec. T > 183 °C; Rf = 0.55
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(BuOH/H2O/AcOH 4:2:1); [α]D = + 336.6 (c = 0.20; DMSO); H NMR (300 MHZ, DMSOꢀ
d6): 1.70–1.82 (1H, m); 1.92–2.06 (1H, m); 2.97 (1H, dd, J = 3.8, 18.5); 3.18 (1H, dd, J = 11.1,
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18.5); 3.58–3.70 (1H, m); 4.60–4.72 (1H, m); 7.02 (2H, J = 8.8); 7.57 (2H, J = 8.8); C NMR
(75 MHz, DMSOꢀd6): 32.66; 37.39; 50.77; 58.37; 83.74; 117.30; 138.29; 142.26; 142.39;
164.31; 170.59; [M+H]+ = 404.1; anal. calcd for C13H14IN3O4: C, 38.73; H, 3.50; N, 10.42;
found: C, 38.98; H, 3.60; N, 10.30.
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(S)-5-((R)-2-amino-2-carboxyethyl)-1-(4-[125I]iodophenyl)-4,5-dihydro-1H-pyrazole-3-
carboxylic acid ([125I]4d).
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To a solution of 14 (0.5 mg, 0.64 µmol) in 0.1 mL of methanol was added 0.001 mL of acetic
acid and 4.6 mCi [125I]NaI solution, followed by Nꢀchlorosuccinimide (0.005 mg, 0.037 µmol) in
methanol. The reaction mixture was kept at room temperature for 20 minutes and then isolated
by radioꢀHPLC (Econosil C18 10 µ, 250 mm × 4.6 mm, H2O/CH3CN/TFA = 70/30/0.1, 1
mL/min). The HPLC eluate at 26 min was collected and evaporated under vacuum. To it
TFA/H2O (95:5, 0.2 mL) was added. The mixture was heated at 40oC for 10 min and isolated by
radioꢀHPLC (Econosil C18 10 µ, 250 mm × 4.6 mm, H2O/CH3CN/TFA = 78/22/0.1, 1 mL/min,
retention time: 17 min) to give [125I]4d. The radiochemical yield was 54% and the specific
activity was 1940 Ci/mmol.
(S)-5-((R)-2-Amino-2-carboxyethyl)-1-p-tolyl-4,5-dihydro-1H-pyrazole-3-carboxylic
acid
(4e). Yield: 62%; yellow solid; m.p. = dec. T > 198 °C; Rf = 0.60 (BuOH/H2O/AcOH 4:2:1);
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[α]D = + 335.00 (c = 0.10; H2O); H NMR (300 MHZ, DMSOꢀd6): 1.65–1.85 (1H, m); 1.90–
2.10 (1H, m); 2.20 (3H, s); 2.95 (1H, dd, J = 4.4, 17.9); 3.15 (1H, dd, J = 11.3, 17.9); 3.35–3.45
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(1H, m); 4.50–4.65 (1H, m); 7.02–7.18 (4H, m); C NMR (75 MHz, DMSOꢀd6): 20.85; 33.18;
37.56; 51.53; 58.66; 114.89; 129.43; 130.22; 141.04; 142.78; 165.08; 170.67; [M+H]+ = 292.1;
anal. calcd for C14H17N3O4: C, 57.72; H, 5.88; N, 14.42; found: C, 58.00; H, 6.01; N, 14.23.
General procedure for the cycloaddition reaction.
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To a solution of (R)ꢀmethyl 2ꢀ(tertꢀbutoxycarbonylamino)pentꢀ4ꢀenoate 8 (1.50 g, 6.54 mmol,
1.0 eq.) in EtOAc (25 mL), the appropriate chloroꢀhydrazone 7aꢀe (9.81 mmol, 1.5 eq.) and solid
NaHCO3 (2.75 g, 32.7 mmol, 5 eq.) were added. The mixture was vigorously stirred for 24 h at
80 °C and the progress of the reaction was followed by TLC (cyclohexane/EtOAc 8:2). Water (5
mL) was added and the organic layer was separated and dried over anhydrous Na2SO4. The crude
material obtained after evaporation of the solvent was purified by column chromatography on
silica gel to give an inseparable mixture of the two diastereoisomers 9aꢀe and 10a-e (Yield: 70ꢀ
90%).
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General procedure for the synthesis of compound 9aꢀe.
a) The mixture of diastereoisomers 9aꢀe and 10aꢀe (4.57 mmol, 1.0 eq.) was treated with a 30%
CH2Cl2 solution of trifluoroacetic acid (3.5 mL, 10 eq.) at 0 °C. The solution was stirred at room
temperature for 3 h and the reaction was followed by TLC (cyclohexane/EtOAc 8:2). A saturated
solution of NaHCO3 (50 mL) and CH2Cl2 (35 mL) were then added and the organic layer was
separated, dried over anhydrous Na2SO4 and evaporated under reduced pressure. The crude
material was purified on silica gel (cyclohexane/EtOAc 3:7) to give the amines 11aꢀe and 12aꢀe
(Yield: 78ꢀ87%).
b) To a stirred solution of the amine 11aꢀe (1.50 mmol, 1.0 eq) in CH2Cl2 (10 mL) cooled at 0
°C, triethylamine (2.25 mmol, 1.5 eq.) and a solution of diꢀtertꢀbutyl dicarbonate (1.95 mmol,
1.3 eq.) in CH2Cl2 (3 mL) were added and the reaction mixture was stirred at room temperature
overnight. The reaction was monitored by TLC (EtOAc). The solvent was evaporated under
reduced pressure and the crude material was purified by column chromatography on silica gel
(cyclohexane/EtOAc 8:2) to give compound 9aꢀe in quantitative yield.
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(S)-Methyl
5-((R)-2-(tert-butoxycarbonylamino)-3-methoxy-3-oxopropyl)-1-(4-
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fluorophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylate (9a). Yield: 87%; yellow oil; Rf = 0.32
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(cyclohexane/EtOAc 7:3); [α]D = + 242.56 (c = 1.00; CHCl3); H NMR (300 MHz, CDCl3):
1.45 (9H, s); 1.82–1.95 (1H, m); 1.95–2.05 (1H, m); 3.10 (1H, dd, J = 4.7, 17.8); 3.40 (1H, dd, J
= 11.3, 17.8); 3.75 (3H, s); 3.90 (3H, s); 4.30–4.42 (1H, m); 4.62 (1H, dddd, J = 1.9, 4.7, 11.3,
11.3); 5.20 (1H, bd, J = 8.4); 6.98–7.06 (2H, m); 7.07–7.15 (2H, m); 13C NMR (75 MHz,
CDCl3): 28.38; 35.09; 37.34; 50.50; 52.32; 52.86; 59.08; 80.73; 116.14 (d, J = 28.8); 116.24;
138.15; 138.68; 156.65; 157.85 (d, J = 300); 163.31; 172.35; [M+H]+ = 424.2; anal. calcd for
C20H26FN3O6: C, 56.73; H, 6.19; N, 9.92; found: C, 56.98; H, 6.31; N, 9.78.
(S)-Methyl
5-((R)-2-(tert-butoxycarbonylamino)-3-methoxy-3-oxopropyl)-1-(4-
0.15
chlorophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylate (9b). yellow oil; Rf
=
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(cyclohexane/EtOAc 8:2); [α]D = + 230.37 (c = 1.00; CHCl3); H NMR (300 MHz, CDCl3):
1.48 (9H, s); 1.80–1.95 (1H, m); 1.95–2.08 (1H, m); 3.10 (1H, dd, J = 4.7, 17.8); 3.40 (1H, dd, J
= 11.3, 17.8); 3.75 (3H, s); 3.87 (3H, s); 4.30–4.42 (1H, m); 4.62 (1H, dddd, J = 2.8, 4.7, 11.3,
11.3); 5.22 (1H, bd, J = 7.5); 7.02–7.12 (2H, m); 7.20–7.28 (2H, m); 13C NMR (75 MHz,
CDCl3): 28.50; 35.00; 37.48; 50.58; 52.49; 52.98; 58.76; 80.79; 116.07; 126.64; 129.47; 139.40;
140.49; 156.04; 163.31; 172.47; [M+H]+ = 440.2; anal. calcd for C20H26ClN3O6: C, 54.61; H,
5.96; N, 9.55; found: C, 54.87; H, 6.12; N, 9.38.
(S)-Methyl
1-(4-bromophenyl)-5-((R)-2-(tert-butoxycarbonylamino)-3-methoxy-3-
oxopropyl)-4,5-dihydro-1H-pyrazole-3-carboxylate (9c). yellow foam; crystallyzed from nꢀ
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hexane; m.p. = 70–72 °C; Rf = 0.25 (cyclohexane/EtOAc 8:2); [α]D = + 268.31 (c = 1.0;
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CHCl3); H NMR (300 MHz, CDCl3): 1.48 (9H, s); 1.80–1.92 (1H, m); 2.02 (1H, ddd, J = 2.4,
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10.6, 13.5); 3.09 (1H, dd, J = 4.7, 17.9); 3.38 (1H, dd, J = 11.7, 17.9); 3.73 (3H, s); 3.87 (3H, s);
4.30–4.42 (1H, m); 4.61 (1H, dddd, J = 2.4, 4.7, 11.7, 11.7); 5.26 (1H, bd, J = 8.5); 6.98–7.04
(2H, m); 7.34–7.41 (2H, m); 13C NMR (75 MHz, CDCl3): 28.53; 35.30; 37.58; 50.56; 52.53;
53.03; 58.69; 80.93; 114.12; 116.43; 132.42; 139.56; 140.91; 155.96; 163.28; 172.33; [M+H]+ =
484.1; anal. calcd for C20H26BrN3O6: C, 49.60; H, 5.41; N, 8.68; found: C, 49.83; H, 5.58; N,
8.52.
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(S)-Methyl 5-((R)-2-(tert-butoxycarbonylamino)-3-methoxy-3-oxopropyl)-1-(4-iodophenyl)-
4,5-dihydro-1H-pyrazole-3-carboxylate (9d). yellow oil; Rf = 0.24 (cyclohexane/EtOAc 8:2);
[α]D20 = + 262.92 (c = 0.25; CHCl3); 1H NMR (300 MHz, CDCl3): 1.48 (9H, s); 1.87 (1H, ddd, J
= 3.5, 10.6, 11.1); 2.02 (1H, ddd, J = 2.4, 10.6, 13.5); 3.09 (1H, dd, J = 4.7, 17.9); 3.38 (1H, dd,
J = 11.1, 17.9); 3.73 (3H, s); 3.87 (3H, s); 4.30–4.42 (1H, m); 4.61 (1H, dddd, J = 2.4, 4.7, 11.1,
11.1); 5.20 (1H, bd, J = 8.5); 6.85–6.95 (2H, m); 7.50–7.60 (2H, m); 13C NMR (75 MHz,
CDCl3): 28.50; 35.25; 37.57; 50.67; 52.50; 52.99; 58.56; 80.90; 83.97; 116.89; 138.28; 139.69;
141.57; 155.97; 163.24; 172.32; [M+H]+ = 532.2; anal. calcd for C20H26IN3O6: C, 45.21; H,
4.93; N, 7.91; found: C, 45.01; H, 4.9381; N, 8.01.
(S)-Methyl
5-((R)-2-(tert-butoxycarbonylamino)-3-methoxy-3-oxopropyl)-1-p-tolyl-4,5-
dihydro-1H-pyrazole-3-carboxylate (9e). yellow foam; Rf = 0.42 (cyclohexane/EtOAc 7:3);
[α]D20 = + 326.90 (c = 1.00; CHCl3); 1H NMR (300 MHz, CDCl3): 1.46 (9H, s); 1.80−1.93 (1H,
m); 2.05 (1H, ddd, J = 2.2, 11.2, 13.5); 2.28 (3H, s); 3.05 (1H, dd, J = 4.9, 17.9); 3.36 (1H, dd, J
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= 11.2, 17.9); 3.70 (3H, s); 3.86 (3H, s); 4.30–4.42 (1H, m); 4.62 (1H, dddd, J = 2.2, 4.7, 11.2,
11.2); 5.20 (1H, bd, J = 8.5); 7.00–7.12 (4H, m); 13C NMR (75 MHz, CDCl3): 20.80, 28.50;
35.11; 37.19; 50.63; 52.30; 52.85; 58.92; 80.69; 115.19; 130.04; 131.36; 137.99; 139.52; 155.44;
163.55; 172.54; [M+H]+ = 520.2; anal. calcd for C21H29N3O6: C, 60.13; H, 6.97; N, 10.02; found:
C, 59.89; H, 6.77; N, 10.20.
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Methyl (S)-5-((R)-2-amino-3-methoxy-3-oxopropyl)-1-(4-[124I]iodophenyl)-4,5-dihydro-1H-
pyrazole-3-carboxylate ([124I]11d).
To a solution of 15 (0.01 mg, 0.014 µmol) in 0.1 mL of methanol was added 0.001 mL of acetic
acid and 4.6 mCi [124I]NaI solution, followed by Nꢀchlorosuccinimide (0.02 mg, 0.15 µmol) in
methanol. The reaction mixture was kept at room temperature for 20 minutes and then dried
under nitrogen. To it TFA/H2O (95:5, 0.2 mL) was added. After 20 min at 45oC, the mixture was
isolated by radioꢀHPLC (Phenomenex C18, 10 µ, 250 mm × 4.6 mm, H2O/CH3CN/TFA =
70/30/0.1, 1 mL/min, retention time: 16 min) to give [124I]11d. The radiochemical yield was 49%
and the specific activity was 13,000 Ci/mmol.
Methyl (S)-5-((R)-2-amino-3-methoxy-3-oxopropyl)-1-(4-[125I]iodophenyl)-4,5-dihydro-1H-
pyrazole-3-carboxylate ([125I]11d).
To a solution of 15 (0.5 mg, 0.72 µmol) in 0.1 mL of methanol was added 0.002 mL of acetic
acid and 1 mCi [125I]NaI solution, followed by Nꢀchlorosuccinimide (0.02 mg, 0.15 µmol) in
methanol. The reaction mixture was kept at room temperature for 20 minutes and then dried
under vacuum. To it TFA/H2O (95:5, 0.2 mL) was added. The mixture was kept at room
temperature for 20 minutes and isolated by radioꢀHPLC (Econosil C18 10 µ, 250 mm × 4.6 mm,
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H2O/CH3CN/TFA = 70/30/0.1, 1 mL/min, retention time: 16 min) to give [125I]11d. The
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radiochemical yield was 58% and the specific activity was 1,000 Ci/mmol.
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Methyl
(S)-5-((R)-2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)-1-(4-
[11C]methylphenyl)-4,5-dihydro-1H-pyrazole-3-carboxylate ([11C]11e).
A reaction vial containing a solution of Pd2(dba)3 (1 mg) and tri(oꢀtolyl)phosphine (1.3 mg) in
0.1 mL DMF was purged with nitrogen for 2 minutes. To it compound 15 (1 mg in 0.1 mL DMF)
was added. [11C]MeI (prepared with GE 11Cꢀmethyl iodide radiosynthesis module and GE
PETtrace cyclotron at Johns Hopkins PET center) was swept by argon flow into the reaction vial.
After the radioactivity reached the plateau, the vial was heated at 90oC for 7 min, 0.25 mL 95%
TFA was added and mixture was heated at 90oC for another 3 min. The reaction was diluted with
0.3 mL water and filtered through a 13 mm Teflon filter (0.45 micron) and purified by HPLC
(Phenomenex Luna C18, 10 ꢀ, 250 mm × 10 mm. H2O/CH3CN/TFA = 70/30/0.1, 8 mL/min,
retention time 9ꢀ10 min) to give [11C]11e. The radiochemical yield was 8ꢀ20 mCi starting from
400ꢀ500 mCi [11C]MeI. Radiochemical purity was >99% and specific radioactivity was 6,000ꢀ
23,000 Ci/mmol.
(S)-tert-Butyl
1-(4-bromophenyl)-5-((R)-3-tert-butoxy-2-(tert-butoxycarbonylamino)-3-
oxopropyl)-4,5-dihydro-1H-pyrazole-3-carboxylate (13)
a) Derivative 9c (290 mg, 0.60 mmol) was dissolved in MeOH (1.8 mL) and treated with 1 N
aqueous NaOH (1.8 mL, 3.0 eq.). The disappearance of the starting material was monitored by
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TLC (cyclohexane/EtOAc 8:2). After evaporation of MeOH, the aqueous layer was washed with
Et2O (1 × 5 mL), made acidic with 2 N aqueous HCl and extracted with EtOAc (3 × 5 mL). The
organic layer was dried over anhydrous Na2SO4 and, after evaporation of the solvent, the diacid
derivative was obtained as a yellow solid (95% yield. b) The diacid intermediate (260 mg, 0.57
mmol) was dissolved in a mixture of dry THF (640 µL) and dry CH2Cl2 (2.40 mL) and tertꢀbutyl
2,2,2ꢀtrichloroacetimidate (5.7 mmol, 1.02 mL) was added. The mixture was stirred overnight at
room temperature. A 5% solution of NaHCO3 (50 mL) was added and the organic layer was
separated. The aqueous phase was extracted with EtOAc (3 × 10 mL). The organic layer was
dried over anhydrous Na2SO4 and, after evaporation of the solvent, the crude was purified by
column chromatography (cyclohexane/EtOAc 9:1). Compound 13 was further purified by
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crystallization from nꢀhexane. Yield: 42%; Rf = 0.59 (cyclohexane/EtOAc 8:2); H NMR (300
MHZ, CDCl3): 1.42 (9H, s); 1.48 (9H, s); 1.58 (9H, s); 1.78–2.05 (2H, m); 3.02 (1H, dd, J = 4.8,
17.8); 3.38 (1H, dd, J = 11.9, 17.8); 4.12–4.25 (1H, m); 4.52–4.65 (1H, m); 5.25 (1H, bd, J =
8.5); 6.98–7.05 (2H, m); 7.32–7.42 (2H, m); anal. calcd for C26H38BrN3O6: C, 54.93; H, 6.74; N,
7.39; found: C, 55.16; H, 6.91; N, 7.27.
tert-Butyl
(S)-5-((R)-3-(tert-butoxy)-2-((tert-butoxycarbonyl)amino)-3-oxopropyl)-1-(4-
(tributylstannyl)phenyl)-4,5-dihydro-1H-pyrazole-3-carboxylate (14).
Compound 13 (0.028 g, 0.049 mmol) and tetrakis(triphenylphosphine)palladium (0.010 g, 0.009
mmol) were placed in an ovenꢀdried, resealable Schlenk tube. Toluene (1 mL) was added via
syringe, followed by hexabutylditin (0.1 mL, 0.2 mmol). The tube was purged with nitrogen for
o
2 minutes and then sealed. Its contents were stirred for 4 hours at 100 C, then cooled to room
temperature and purified by flash chromatography (silica gel, EtOAc: hexanes = 1 : 4) to afford
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14 (0.021 g, 55%). 1H NMR (400 MHz, CDCl3) δ 7.34 (2H, d, J = 8.0 Hz), 7.11 (2H, d, J = 8.0
Hz), 5.30 (1H, m), 4.59 (1H, m), 4.21 (1H, m), 3.31 (1H, m), 3.01 (1H, m), 2.01 (1H, m), 1.85
(1H, m), 1.43ꢀ1.54 (33H, m), 1.23ꢀ1.29 (6H, m), 0.97ꢀ1.02 (6H, m), 0.84ꢀ0.88 (9H, m).
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Methyl
(S)-5-((R)-2-((tert-butoxycarbonyl)amino)-3-methoxy-3-oxopropyl)-1-(4-
(tributylstannyl)phenyl)-4,5-dihydro-1H-pyrazole-3-carboxylate (15).
Compound 9c (0.050 g, 0.103 mmol) and tetrakis(triphenylphosphine)palladium (0.010 g, 0.009
mmol) were placed in an ovenꢀdried, resealable Schlenk tube. Toluene (2 mL) was added via
syringe, followed by hexabutylditin (0.3 mL, 0.6 mmol). The tube was purged with nitrogen for
°
2 min and then sealed. Its contents were stirred overnight at 95 C, then cooled to room
temperature and purified by flash chromatography (silica gel, EtOAc: hexanes = 1:3) to afford 15
(0.024 g, 33%). 1H NMR (400 MHz, CDCl3) δ 7.34 (2H, d, J = 8.0 Hz), 7.10 (2H, d, J = 8.0 Hz),
5.20 (1H, m), 4.64 (1H, m), 4.37 (1H, m), 3.85 (3H, s), 3.71 (3H, s), 3.36 (1H, m), 3.07 (1H, m),
2.06 (1H, m), 1.85 (1H, m), 1.44ꢀ1.58 (15H, m), 1.29ꢀ1.39 (6H, m), 0.98ꢀ1.02 (6H, m), 0.84ꢀ0.88
(9H, m).
Animals. All animal studies were performed in compliance with a Johns Hopkins Animal Care
and Use Committee approved protocol. Male CDꢀ1 mice (7 weeks of age) were purchased from
Charles River Laboratories (Frederick, MD) and were housed for several weeks in a specific
pathogen free facility under a 12/12 light/dark cycle with free access to food and water ad
libitum. Female athymic nude mice were purchased from Jackson Labs (Bar Harbor, ME) and
housed under the same conditions. Rodent chow is Teklad 8604 (Harlan Labs, Frederick, MD).
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Autoradiography. Athymic nude mice (n = 1 per compound) were injected with either 47 MBq
(1.27 mCi, 1,940 Ci/mmol) of [125I]4d for a 60 min conscious uptake or 9.1 MBq (246 ꢀCi,
1,000 Ci/mmol) of [125I]11d for a 40 min conscious uptake. No anesthetics were used. Following
the indicated uptake times, the mice were sacrificed by cervical dislocation and their brains were
rapidly harvested and frozen over dry ice. The brains were then sectioned using a cryotome (HM
Microm 550, Microm Intl. GmbH, Waldorf, Germany) to 20 µm thickness and adhered to
charged glass slides (VWR, Radnor, PA) before exposing them to xꢀray film (Kodak Biomax
XR, Sigma, St. Louis, MO). Brain sections containing compound [125I]4d were exposed for 18 h
while sections containing compound [125I]11d were exposed for 24 h. Films were developed
using a Kodak XꢀOꢀMat film processor (Carestream, Rochester, NY). Exposures were digitized
and displayed using an MCID Core system (MCID, Nottingham, UK) using the manufacturer’s
software.
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Rodent biodistribution. [124I]11d: Four male CDꢀ1 mice were each injected intravenously with
1.85 MBq (50 µCi, 1,000 Ci/mmol) of radiotracer in 100 µL of PBS. They mice underwent a 75
min anestheticꢀfree uptake period prior to sacrifice by cervical dislocation. Selected tissues
(blood, muscle, cerebellum, olfactory bulb, hypothalamus, hippocampus, caudateꢀputamen,
frontal cortex, cortex, brain stem, thalamus, superior colliculus and rest of brain) were rapidly
dissected, weighed and counted on a gamma counter (CS Compugamma 1282, LKB Wallac,
Waverly, Vic) alongside diluted standard doses to obtain a percent injected dose per gram of wet
tissue (% ID/g). Tissue uptake per region was averaged and graphed with standard deviation as
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indicated. [11C]11e: kinetic biodistribution was carried out at 15, 30, 45 and 60 min postꢀ
injection (n = 4 mice per time point) following the I.V. injection of 3.7 MBq (100 µCi, 1,400
Ci/mmol) of radiotracer in 100 µL of PBS. Similarly, for the autoblockade study, 3.7 MBq
(11,500 Ci/mmol) of radiotracer was injected alone or in combination with 4.5 mg/kg of
unlabeled 11e (n = 4/ group) in 100 µL of PBS. Time points for the blockade study were 60 min
(tracerꢀonly and blocked) and 75 min (tracerꢀonly). Tissues harvested and procedures were
identical to those listed for [124I]11d.
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In vivo PETꢀCT imaging. Two female athymic nude mice underwent dynamic positron emission
tomography (PET) imaging. The mice were maintained under anesthesia with 2.5% isoflurane in
oxygen (2L/min) and positioned sideꢀbyꢀside in a small animal SuperArgus PET/CT system
(Sedecal, Madrid, Spain). One mouse was administered 8.14 MBq (220 ꢂCi) of ([124I]11d)
intravenously as a bolus. The other mouse received the same dose in addition to 150 mg/kg of
ketamine. Dynamic PET imaging started simultaneously with dose administration and proceeded
for 2 h, with the imaging field of view centered over the brain. A CT scan utilizing 360
projections at 60 keV was performed for anatomic coꢀregistration purposes. Dynamic PET data
were reconstructed using the manufacturerꢀprovided 2D OSEM algorithm21 and CT data were
reconstructed with a manufacturerꢀprovided standard filtered backꢀprojection method. The PET
reconstructed images were first coꢀregistered with the CT using anatomic landmarks such as
anterior eye socket to Harderian gland PET signal and displayed using AMIDE
(http://amide.sourceforge.net/). Slices and 3D reconstructions were evaluated for brain uptake of
radiotracer.
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